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Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2–4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein–protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance. CRISPR screening and protein–protein interaction networks identify components and mechanisms of nutrient-dependent mTORC1 signalling in regulatory T cells and reveal how mTORC1 integrates immunological cues and nutrient signals for adaptive immunity.

The detection of microorganism-associated ligands by plant cells activates a signalling cascade in which the kinase BIK1 is monoubiquinated, released from the FLS2-BAK1 complex, and internalized by endocytosis. Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens(1-3). Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants(4). The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.

Sleep and wake have global effects on brain physiology, from molecular changes1–4 and neuronal activities to synaptic plasticity3–7. Sleep–wake homeostasis is maintained by the generation of a sleep need that accumulates during waking and dissipates during sleep8–11. Here we investigate the molecular basis of sleep need using quantitative phosphoproteomic analysis of the sleep-deprived and Sleepy mouse models of increased sleep need. Sleep deprivation induces cumulative phosphorylation of the brain proteome, which dissipates during sleep. Sleepy mice, owing to a gain-of-function mutation in the Sik3 gene 12 , have a constitutively high sleep need despite increased sleep amount. The brain proteome of these mice exhibits hyperphosphorylation, similar to that seen in the brain of sleep-deprived mice. Comparison of the two models identifies 80 mostly synaptic sleep-need-index phosphoproteins (SNIPPs), in which phosphorylation states closely parallel changes of sleep need. SLEEPY, the mutant SIK3 protein, preferentially associates with and phosphorylates SNIPPs. Inhibition of SIK3 activity reduces phosphorylation of SNIPPs and slow wave activity during non-rapid-eye-movement sleep, the best known measurable index of sleep need, in both Sleepy mice and sleep-deprived wild-type mice. Our results suggest that phosphorylation of SNIPPs accumulates and dissipates in relation to sleep need, and therefore SNIPP phosphorylation is a molecular signature of sleep need. Whereas waking encodes memories by potentiating synapses, sleep consolidates memories and restores synaptic homeostasis by globally downscaling excitatory synapses4–6. Thus, the phosphorylation–dephosphorylation cycle of SNIPPs may represent a major regulatory mechanism that underlies both synaptic homeostasis and sleep–wake homeostasis.

Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours 1 . Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified 2 , the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α− dendritic cells. Dendritic cell-specific deletion of Mst1/2—but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling—disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α− dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α− dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets. A data-driven analysis helps to identify specific roles of the Hippo signalling kinases Mst1 and Mst2 in integrating metabolic activity and cytokine signalling in dendritic cells, and thereby orchestrating immune cell function.

G protein-coupled receptors (GPCRs) are transmembrane proteins that modulate physiology across diverse tissues in response to extracellular signals. GPCR signalling can differ due to variation in the sequence (e.g. polymorphisms) or in the expression of receptors in different tissues. The resulting differences in response are an important source of physiological signalling bias. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms that can have distinct patterns of expression in human tissues. Here, we report the findings from a comprehensive study, integrating data from human tissue-level transcriptomes, GPCR sequences and structures, functional annotations, proteomics, single-cell RNA sequencing, population-wide genetic association studies, and pharmacological experiments. Our results show how a single GPCR gene can diversify into multiple isoforms with distinct structural and signalling properties, and how unique combinations of these isoforms can be expressed in different human tissues, contributing to differences in physiological signalling. Based on their structural changes and expression patterns, some of the detected isoforms may also influence drug response and represent new drug targets with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling, in which one considers how the combinatorial expression of receptor isoforms in a specific system (i.e. a particular cell type, tissue, or organism) collectively impacts receptor signalling. These observations pave the way for understanding the impact of isoform variation on GPCR signalling response and have implications for exploiting such variation as a source of GPCR selectivity in drug development.

Argonaute proteins are a part of a larger RNA interference (RNAi) complex, RISC, in which they mediate cleavage of target mRNAs. In this study, Shi and colleagues show that Ago2 interacts with and is hydroxylated by type I collagen prolyl-4-hydroxylase. When this activity is depleted in human or mouse cells, the level of Ago2 is reduced and siRISC activity mediated by the let-7 miRNA is affected. These results suggest that hydroxylation of Ago2, by affecting its stability, can influence the efficiency of RNAi. Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference1. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies1,2. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the α-(P4H-α(I)) and β-(P4H-β) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-α(I) or P4H-β by short hairpin RNA and P4H-α(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.